In this study, we have evaluated the potential therapeutic benefit of using a small molecule TGFβ signaling inhibitor with doxorubicin in animal models of breast cancer. Doxorubicin, which is a frequently used chemotherapeutic agent against metastatic breast cancer, has been reported to induce acute and chronic toxicities and immunosuppression. In addition, we found in our study that doxorubicin can activate TGFβ signaling in cancer cells, which has the potential to accelerate malignant tumor progression. Thus, novel treatment options are needed to reduce dosages of doxorubicin and consequently unwanted toxicities while enhancing its therapeutic efficacy in treating metastatic cancer.
An elevated plasma level of TGFβ has been correlated to the progression of various types of malignancies including metastatic breast cancer progression 
and survival 
. Persistent high levels of circulating TGFβ predicted early metastatic recurrences in the liver of patients with colorectal cancer after curative resection 
. In addition to the carcinogenesis-associated upregulation of TGFβ expression, several chemotherapeutic agents including doxorubicin were shown to stimulate TGFβ1 expression and secretion from xenograft tumors 
. Surprisingly, in a recent study, it was shown that doxorubicin treatment caused increased circulating levels of TGFβ as well as increased circulating tumor cells and lung metastases in the MMTV/PyVmT transgenic animal model 
. We have observed that the presence of doxorubicin induced the migration and invasion of murine 4T1 and human MDA-MB-231 breast cancer cells similar to the TGFβ1 treated cells in a Boyden chamber assay. Further investigation revealed that doxorubicin stimulated the phosphorylation of Smad2 and Smad3 in both 4T1 and MDA-MB-231 cells. Consistent with the stimulation of Smad2 and Smad3 phosphorylation, doxorubicin also stimulated the TGFβ-responsive PAI-1 promoter activity in a dose dependent manner. Our results strongly indicate that although doxorubicin can induce cytostatic and/or cytocidal effects, it can also induce cancer cell migration/invasion by activating TGFβ pathway.
EMT is a latent embryonic program by which some cells within a tumor during cancer progression can reactivate mesenchymal traits to disseminate to metastatic sites 
. Induction of EMT by TGFβ has been shown to increase motility and invasiveness via the disassembly of cell-to-cell contacts, loss of cell polarity and significant cytoskeletal reorganization in normal and malignant mammary epithelial cell types. We observed in the murine breast cancer 4T1 cell model that TGFβ and surprisingly also doxorubicin can induce EMT as evidenced by the mesenchymal morphology, reorganization of the actin cytoskeleton and nuclear localization of the embryonic transcription factor Snail.
Emerging evidence has shown that certain type of tumors including breast tumors contain a subset of cells that initiate and propagate the tumors with high efficiency. These cells are referred as cancer stem cells (CSCs) and often exhibit phenotypes resembling normal stem cell properties such as expression of stem cell markers, self renewal and multipotency 
. Metastatic breast cancer cells are notorious for their ability to survive treatment and regenerate tumors. One important feature of CSCs is their resistance to the cytotoxicity of varous chemotherapeutic agents because of their relatively high expression levels of multiple drug resistance transporters such as ABCB1 (MDR1/PGP, P-glycoprotein) and ABCG2 (MXR/BCRP) 
. Thus, chemotherapeutics might spare CSC's and allow recurrence of more aggressive tumors. Recent data showed that immortalized human and murine breast cancer cell lines contain stem-like cells similar to CSCs isolated from primary tumor tissue with identical cell surface markers, clonogenic and sphere formation potential, and in vivo functional transplantation properties 
. The fact that EMT can generate stem-like cancer cells 
suggests that therapeutic-resistant cancer stem cells may be the result of either the transformation of normal stem cells or via the induction of EMT in more differentiated cancer cells. Indeed, we found that treatment with TGFβ or doxorubicin increased the number of 4T1 cells expressing stem cell marker Sca-1 and the drug resistant transporter protein MDR1, and with the stem cell property of self-renewal as indicated by mammosphere formation. The 4T1 cells pre-treated with TGFβ1 for 7 days showed resistance to the doxorubicin-induced growth inhibition.
Our results indicate that by activating TGFβ signaling, doxorubicin has the ability to induce EMT and generate tumor cells with stem cell phenotypes. The EMT program is often considered a transient and reversible process. Studies of embryonic development have showed that mesenchymal cells of mesoderm can give rise to epithelial organs, including the kidney and ovary, by undergoing the reverse EMT 
. The finding that EMT plays a role in the development of cancer stem cells suggests that therapies blocking or reversing the EMT program may sensitize resistant cancer stem cells to conventional cancer therapies. We have evaluated the ability of a small molecule TGFβ inhibitor, TβRI-KI, to block the EMT process and subsequent cancer stem cell formation by TGFβ1 and doxorubicin treatment in 4T1 cells in vitro. We found that blockade of TGFβ signaling significantly reversed the TGFβ1- and doxorubicin-mediated EMT process. Restoration of the epithelial phenotypes of the cytoskeleton organization and cytoplasmic localization of the EMT transcription factor Snail was observed in the presence of the inhibitor. The treatment of TβRI-KI also inhibited TGFβ1- and doxorubicin-mediated generation of cells with stem cell phenotypes including the expression of MDR1 and the formation of mammospheres.
Thus, all our in vitro experiments indicated potential therapeutic benefits of using TβRI-KI to antagonize the doxorubicin-induced TGFβ signaling, which has the potential to induce EMT and to generate drug resistant stem-like cancer cells. We designed in vivo experiments utilizing murine breast cancer 4T1 cells and human breast cancer MDA-MB-231 cells to study the synergistic anti-tumor effect of the systemic administration of both doxorubicin and TβRI-KI in comparison to single-agent treatments. Our studies revealed that while treatment with either agent alone moderately inhibited tumor growth in both xenograft breast cancer models, the combination of both agents was clearly much more effective in inhibiting tumor growth. In fact, a 50% reduction of doxorubicin dose was sufficient to produce the same tumor growth inhibition as the full dose when TβRI-KI was added to the treatment suggesting that the combination with TGFβ antagonists may be an effective strategy in reducing doxorubicin dosage and its unwanted side-effects.
Lung metastasis is prevalent among the patients with metastatic breast cancer 
. About 60% to 74% of patients who die of breast carcinoma have lung metastasis 
. In several animal models, TGFβ has been shown to promote lung metastasis 
. Since Doxorubicin is widely used as an adjuvant chemotherapeutic agent in metastatic breast cancer with limited response rate (4) and enhanced lung metastasis in an animal model 
, we also examined the lungs of the animals for the presence of metastatic colonies in the placebo and treatment groups. We observed moderate reduction of lung metastasis after doxorubicin treatment which was significantly improved by combinational therapy with TβRI-KI in the murine 4T1 xenograft model. In the human breast cancer xenograft model, our results indicated that doxorubicin alone can stimulate lung metastasis with larger metastatic colonies which can be effectively blocked by the simultaneous treatment with TβRI-KI. Since smaller sized pulmonary metastasis has been shown to be significantly associated with longer survival 
, our results suggest that administration of TβRI-KI along with doxorubicin may have the potential to improve the survival rate of doxorubicin-treated metastatic breast cancer patients.
Metastases to bone are frequently seen pathological lesions in advanced breast cancer, which causes debilitating skeletal complications such as osteolysis, intractable bone pain and pathologic fractures 
. Most importantly, once breast tumor cells metastasize to bone marrow, mortality increases to 70% in comparison to 40% in the breast cancer patients with no tumor cells in their bone marrow 
. Therefore, prevention and treatment of bone metastasis is important for the improvement of survival in breast cancer patients. In our study, we observed that mice bearing orthotopic 4T1 tumors showed an increased incidence of spontaneous bone metastasis as well as greater bone tumor burden after doxorubicin treatment in comparison to the placebo treatment. Both incidence of tumor cells and tumor volume in the bone marrow were reduced remarkably after simultaneous treatment with TβRI-KI even though TβRI-KI treatment alone was only moderately effective in reducing bone metastasis incidence and tumor burden. Thus, the combination therapy appears to produce a synergistic inhibition of bone metastasis in this model system.
In summary, our study suggests that doxorubicin at certain dosages can promote breast cancer invasion and metastasis in part through its ability to activate TGFβ signaling, which in turn generates stem-like cancer cells with increased drug resistance through an EMT process. Our study also suggests that administration of TGFβ antagonists during chemotherapy may sensitize carcinoma cells, in part through the inhibition of EMT, to the anti-tumor and anti-metastatic activities of chemotherapy resulting in increased therapeutic efficacy and reduced dosage of chemotherapeutics and their unwanted side effects.